Yongzheng Wu

Aspergillus fumigatus-induced Interleukin-8 Synthesis by Respiratory Epithelial Cells Is Controlled by the Phosphatidylinositol 3-Kinase, p38 MAPK, and ERK1/2 Pathways and Not by the Toll-like Receptor-MyD88 Pathway

Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-κB-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-κB activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis.

Anthrax Lethal Toxin Impairs IL-8 Expression in Epithelial Cells through Inhibition of Histone H3 Modification

Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis, the etiological agent of anthrax, whose pulmonary form is fatal in the absence of treatment. Inflammatory response is a key process of host defense against invading pathogens. We report here that intranasal instillation of a B. anthracis strain bearing inactive LT stimulates cytokine production and polymorphonuclear (PMN) neutrophils recruitment in lungs. These responses are repressed by a prior instillation of an LT preparation. In contrast, instillation of a B. anthracis strain expressing active LT represses lung inflammation. The inhibitory effects of LT on cytokine production are also observed in vitro using mouse and human pulmonary epithelial cells. These effects are associated with an alteration of ERK and p38-MAPK phosphorylation, but not JNK phosphorylation. We demonstrate that although NF-κB is essential for IL-8 expression, LT downregulates this expression without interfering with NF-κB activation in epithelial cells. Histone modifications are known to induce chromatin remodelling, thereby enhancing NF-κB binding on promoters of a subset of genes involved in immune response. We show that LT selectively prevents histone H3 phosphorylation at Ser 10 and recruitment of the p65 subunit of NF-κB at the IL-8 and KC promoters. Our results suggest that B. anthracis represses the immune response, in part by altering chromatin accessibility of IL-8 promoter to NF-κB in epithelial cells. This epigenetic reprogramming, in addition to previously reported effects of LT, may represent an efficient strategy used by B. anthracis for invading the host.

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host.

Type-IIA secreted phospholipase A(2) (sPLA(2)-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA(2)-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA(2)-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA(2)-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA(2)-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA(2)-IIA. Transgenic mice over-expressing human sPLA(2)-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA(2)-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA(2)-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA(2)-IIA, indicating that instilled sPLA(2)-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA(2)-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.

Critical role of cytosolic phospholipase A2α in bronchial mucus hypersecretion in CFTR-deficient mice

Cystic fibrosis (CF) is due to mutations in the CF transmembrane conductance regulator gene CFTR. CF is characterised by mucus dehydration, chronic bacterial infection and inflammation, and increased levels of cytosolic phospholipase A2&agr; (cPLA2&agr;) products in airways. We aimed to examine the role of cPLA2&agr; in the modulation of mucus production and inflammation in CFTR-deficient mice and epithelial cells. Mucus production was assessed using histological analyses, immuno-histochemistry and MUC5AC ELISA. cPLA2&agr; activation was measured using an enzymatic assay and lung inflammation determined by histological analyses and polymorphonuclear neutrophil counts in bronchoalveolar lavages. In lungs from Cftr-/- mice, lipopolysaccharide induced mucus overproduction and MUC5AC expression associated with an increased cPLA2&agr; activity. Mucus overproduction was mimicked by instillation of the cPLA2&agr; product arachidonic acid, and abolished by either a cPLA2&agr; null mutation or pharmacological inhibition. An increased cPLA2&agr; activity was observed in bronchial explants from CF patients. CFTR silencing induced cPLA2&agr; activation and MUC5AC expression in bronchial human epithelial cells. This expression was enhanced by arachidonic acid and reduced by cPLA2&agr; inhibition. However, inhibition of CFTR chloride transport function had no effect on MUC5AC expression. Reduction of CFTR expression increased cPLA2&agr; activity. This led to an enhanced mucus production in airway epithelia independent of CFTR chloride transport function. cPLA2&agr; represents a suitable new target for therapeutic intervention in CF.

Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A2 expression through MAPK/NF-κB inactivation

Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A(2) (sPLA(2)-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA(2)-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA(2)-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA(2)-IIA expression in AMs indicating that these kinases play a role in sPLA(2)-IIA expression. LT inhibited IL-1beta-induced p38 phosphorylation as well as sPLA(2)-IIA promoter activity in CHO cells. Inhibition of sPLA(2)-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1beta-induced NF-kappaB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IkappaBalpha degradation or NF-kappaB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA(2)-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA(2)-IIA expression is induced via a sequential MAPK-NF-kappaB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-kappaB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.

Secreted Group IIA Phospholipase A2 Protects Humans Against the Group B Streptococcus: Experimental and Clinical Evidence

Group B streptococcus (GBS) is a leading neonatal pathogen and a growing cause of invasive disease in the elderly, with clinical manifestations such as pneumonia and sepsis. Despite its clinical importance, little is known about innate immunity against GBS in humans. Here, we analyze the role of human group IIA secreted phospholipase A2 (sPLA2-IIA), a bactericidal enzyme induced during acute inflammation, in innate immunity against GBS. We show that clinical GBS isolates are highly sensitive to killing by sPLA2-IIA but not by human antimicrobial peptides. Using transgenic mice that express human sPLA2-IIA, we demonstrate that this enzyme is crucial for host protection against systemic infection and lung challenge by GBS. We found that acute sera from humans diagnosed with invasive GBS disease contain increased levels of sPLA2-IIA compared with normal sera from healthy individuals, indicating that GBS induces an sPLA2-IIA response in blood during human infection. We demonstrate that clinically relevant GBS strains are rapidly killed in these acute sera. We also demonstrate that the bactericidal effect is entirely due to sPLA2-IIA, showing that sPLA2-IIA might represent an important component of humoral innate immunity against GBS. Our data provide experimental and clinical evidence that sPLA2-IIA protects humans against GBS infections.

A Novel Bacterial Resistance Mechanism against Human Group IIA-Secreted Phospholipase A2: Role of Streptococcus pyogenes Sortase A

Human group IIA-secreted phospholipase A2 (sPLA2-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA2-IIA. Analysis of a set of clinical isolates demonstrated that an ∼500-fold higher concentration of sPLA2-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA2-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA2-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (∼30-fold) to sPLA2-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA2-IIA. Importantly, using transgenic mice expressing human sPLA2-IIA, we showed that the sortase A-mediated sPLA2-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA2-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA2-IIA and we showed that sPLA2-IIA contributes to the host defense against S. pyogenes infection.

Toll-Like Receptor 9 Deficiency Protects Mice against Pseudomonas aeruginosa Lung Infection

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. While a number of studies have demonstrated the roles of TLR2, TLR4 and TLR5 in host defense againt P. aeruginosa infection, the implication of TLR9 in this process has been overlooked. Here, we show that P. aeruginosa DNA stimulates the inflammatory response through TLR9 pathway in both a cell line and primary alveolar macrophages (AMs). This activation requires asparagine endopeptidase- and endosomal acidification. Interestingly, TLR9-/- mice resisted to lethal lung infection by P. aeruginosa, compared to WT C57BL/6 mice. The resistance of TLR9-/- mice to P. aeruginosa infection was associated with: (i) a higher ability of TLR9-/- AMs to kill P. aeruginosa; (ii) a rapid increase in the pro-inflammatory cytokines such as TNFα, IL-1β and IL-6 production; and (iii) an increase in nitric oxide (NO) production and inductible NO synthase expression in AMs. In addition, inhibition of both IL-1β and NO production resulted in a significant decrease of P. aeruginosa clearance by AMs. Altogether these results indicate that TLR9 plays a detrimental role in pulmonary host defense toward P. aeruginosa by reducing the AMs clearance activity and production of IL-1β and NO necessary for bacteria killing.

Neisseria meningitidis pili induce type‐IIA phospholipase A2 expression in alveolar macrophages

Induction of type‐IIA secreted phospholipase A2 (sPLA2‐IIA) expression by bacterial components other than lipopolysaccharide has not been previously investigated. Here, we show that exposure of alveolar macrophages (AM) to Neisseria meningitidis or its lipooligosaccharide (LOS) induced sPLA2‐IIA synthesis. However, N. meningitidis mutant devoid of LOS did not abolish this effect. In addition, a pili‐defective mutant exhibited significantly lower capacity to stimulate sPLA2‐IIA synthesis than the wild‐type strain. Moreover, pili isolated from a LOS‐defective strain induced sPLA2‐IIA expression and nuclear factor kappa B (NF‐κB) activation. These data suggest that pili are potent inducers of sPLA2‐IIA expression by AM, through a NF‐κB‐dependent process.

CFTR dysfunction induces vascular endothelial growth factor synthesis in airway epithelium

Peribronchial angiogenesis may occur in cystic fibrosis and vascular endothelial growth factor (VEGF)-A regulates angiogenesis in airways. Peribronchial vascularity and VEGF-A expression were examined using immunocytochemistry and morphometric analysis in lung sections obtained in 10 cystic fibrosis patients at transplantation versus 10 control nonsmokers, and in two strains of Cftr-deficient mice versus wild-type littermates. Airway epithelial NCI-H292 cells and primary cultures of noncystic fibrosis human airway epithelial cells were treated with cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors (CFTR-inh172 or PPQ-102) or transfected with a CFTR small interfering (si)RNA with or without a selective epidermal growth factor receptor tyrosine kinase inhibitor. Concentrations of VEGF-A and phosphorylated epidermal growth factor receptor were measured by ELISA. Peribronchial vascularity was increased in cystic fibrosis patients, but not in Cftr-deficient mice. VEGF-A immunostaining was localised to airway epithelium and was increased in cystic fibrosis patients and in Cftr-deficient mice. In cultured airway epithelial cells, treatment with CFTR inhibitors or transfection with CFTR siRNA induced a twofold increase in VEGF-A production. CFTR inhibitors triggered epidermal growth factor receptor phosphorylation that was required for VEGF-A synthesis. Cystic fibrosis airways at transplantation showed increased peribronchial vascularity and epithelial VEGF-A expression. CFTR dysfunction triggered epithelial synthesis of VEGF-A, which may contribute to vascular remodelling. CFTR dysfunction triggers epithelial synthesis of VEGF-A, which may contribute to vascular remodelling http://ow.ly/p7ebY